Butyrate-inducible elements in the human γ-globin promoter

Experimental Hematology 28 (2000) 283–293

Butyrate-inducible elements in the human ␥-globin promoter Betty S. Pacea,b, Ying-Ru Chena, Amy Thompsona, and Steven R. Goodmana a

Departments of Structural and Cellular Biology, and bPediatrics, University of South Alabama, Mobile, Ala., USA (Received 13 January 1999; revised 17 November 1999; accepted 23 November 1999)

Objective. Several agents including hydroxyurea, erythropoietin and butyric acid have been shown to reactivate ␥ gene expression during adult stage development by unknown molecular mechanisms. In addition to inhibiting the enzyme histone deacetylase, butyrate may modulate transcription factor binding to specific DNA sequences defined as butyrate response elements (BREs). The purpose of this study was to identify promoter sequences involved in ␥ gene activation by butyrate using truncation mutants in stable cell lines. Materials and Methods. A detailed analysis of A␥ gene activation in the presence of ␣-aminobutyric acid and sodium butyrate was completed in stable mouse erythroleukemia (MEL) cell pools established with seven A␥ promoter truncation mutants. Functional studies were performed in a transient assay system followed by gel mobility shift assays to define protein binding patterns and to demonstrate transcription factor interactions in the ␥ promoter BRE. Results. A␥ promoter analysis in stable MEL cell pools revealed BREs between nucleotide141 and ⴚ201, and nucleotide-822 and -893 (␥BRE). The ␥BRE required the minimal A␥ promoter (ⴚ201 to ⴙ36) to stimulate gene expression. We observed a 6.1-fold (p ⬍ 0.05) increase in CAT activity for the minimal A␥ promoter alone compared with an 11.5-fold (p ⬍ 0.05) increase when the ␥ promoter was combined with the ⴚ822 to ⴚ893 fragment. Protein binding studies demonstrated altered protein-DNA interactions in the ␥BRE after butyrate induction. The pattern for binding observed suggest both negative- and positive-acting transcription factors may interact in this region. Conclusion. The data supports the ⴚ822 to ⴚ893 region as a DNA regulatory element that contributes to A␥ gene inducibility by butyrate. © 2000 International Society for Experimental Hematology. Published by Elsevier Science Inc. Keywords: Butyrate response element—Fetal hemoglobin—Mouse erythroleukemia cells—␥-Globin

Introduction The nuclear transacting factors and cis-regulatory elements involved in the switch from ␥- to ␤-globin gene expression during fetal development and erythroid maturation have been partially defined [1]. Several pharmacologic agents are known to reverse the switch resulting in ␥ gene reactivation and fetal hemoglobin (Hb F) production. These agents include cytotoxic compounds (didox, hydroxyurea, and 5-azacytidine) [2–6], cytokines (erythropoietin) [7], and butyric acid and its various analogs (␣-amino butyric acid and phenylbutyrate) [8,9]. Cytotoxic agents most likely induce fetal hemoglobin production by producing rapid erythroid regener-

Offprint requests to: Betty S. Pace, M.D., University of South Alabama, Department of Structural and Cellular Biology, 307 University Blvd., MSB 2042, Mobile, AL 36688-0002 USA; E-mail: [email protected]

ation following cytoreduction [10,11]. Cytokines may act by direct or indirect gene activation through signal transduction pathways. Butyrate, however, is known to have several effects in culture and in vivo [12], such as cellular growth arrest in the G1 phase [13], protein synthesis induction, changes in cell morphology and cytoskeleton [14], and induction of differentiation in erythroleukemia cell lines [15,16]. Sodium butyrate (NaB) also induces globin gene expression and Hb F production in colonies derived from neonatal cells [17], adult baboons, or patients with sickle cell anemia [18]. More recently, butyric acid has been shown to inhibit the ␥- to ␤-globin switch in human fetal erythroid cells fused with mouse erythroleukemia (MEL) cells [19]. The mechanism whereby butyrate compounds inhibit or reverse hemoglobin switching has been the focus of several investigations. Perrine et al. [20] demonstrated a delay in fetal hemoglobin switching in infants of diabetic mothers due

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to increased ␣-amino butyric acid (␣-ABA) levels in blood. Several studies support the known ability of NaB to modify histone acetylation levels [21,22] as a possible mechanism for ␥-globin gene reactivation and Hb F production. Previous work from our laboratory [23] demonstrated the requirement for ␥-gene preactivation and hypomethylation of the promoter to achieve increased ␥-gene expression by butyrate in adult erythroblasts. Several investigators have demonstrated that NaB treatment results in modified nuclear transactivating factor interactions with specific DNA sequences. Such sequences defined as butyrate response elements (BREs) have been identified in the promoter of the embryonic chicken ␳ gene [24], the histone H1 gene [25], and several other genes [26–28]. Recently, BREs have been localized to the duplicated CCAAT box [29] and the stage selector element [30] in the ␥-globin promoter. Previous studies in our laboratory to localize BREs in the A␥-gene promoter were performed in transgenic mice carrying a 2.5-kb cassette containing four DNaseI hypersensitive sites from the ␤-globin locus control region (␮LCR) linked to the human A␥ gene [31]. The level of A␥ mRNA was increased sixfold after ␣-ABA treatment in this transgenic line. Four additional transgenic lines were analyzed and potential BREs identified between nucleotide ⫺141 to ⫺201 and ⫺730 and ⫺1350 in the A␥ globin promoter [31]. In this study, a de-

tailed truncation analysis of the A␥ promoter was completed in stable MEL cell pools. Two butyrate responsive sequences were identified in the A␥ promoter in vitro. Transient transfection experiments confirmed butyrate inducibility for the minimal A␥ promoter (⫺201 to ⫹36) as previously shown by others [29, 30]. In addition, we observed enhanced gene expression when the ⫺822 to ⫺893 (␥BRE) sequence was combined with the minimal ␥ promoter. Nuclear protein binding studies showed altered protein-DNA interactions in the ␥BRE after butyrate induction. These data support the ⫺822 to ⫺893 region as a DNA regulatory element capable of binding nuclear transacting factors altered by butyrate.

Material and methods Plasmid constructs A diagram illustrating the truncations of the A␥ promoter used to make the recombinant DNA constructs is shown in Fig. 1A. Seven constructs containing the A␥ gene linked to a 2.5-kb ␮LCR cassette composed of core elements from the four erythroid-specific hypersensitive sites located in the ␤-globin LCR were analyzed [32]. The constructs have a 3⬘ end at the HindIII position 530-bp downstream of the polyadenylation site (⫹1950) for the human A␥ gene, while differing at the 5⬘ end. Five of the seven constructs have been previously described [32, 33]. In brief, the 5⬘ end of the

Figure 1. (A) Schematic diagram of the constructs used to establish the MEL cell pools. All constructs carry a 2.5-kb cassette containing the core sequences from 5⬘ hypersensitive sites 1–4 located in the ␤ globin locus control region (␮LCR) and the A␥ gene with 3⬘ ends at nucleotide ⫹1950 relative to the cap site and 5⬘ ends at the positions indicated. The length of the ␥ promoter fragments and size of the ␮LCR cassette are not drawn to scale. (B) RNase protection analysis for A␥ gene activation by ␣-ABA in MEL cell pools. Total RNA (200ng) was hybridized with radiolabeled RNA probes specific for human A␥, murine ␣, and actin (see Material and Methods section). Each panel shows a representative gel for the pattern of A␥ gene expression in the uninduced (U), ␣-ABA-treated (A) or hexamethyl bisacetamide-induced (H) states for the stable MEL cell pools. Note the significant increase in ␥ mRNA production at baseline for the ␮LCR-201A␥ pools (lane 4) and the induction of ␥ gene activity in the presence of ␣-ABA for the ␮LCR-893A␥ pool (lanes 16 and 17).

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␮LCR-141A␥ construct is in the NcoI site at position ⫺141. The ␥ promoter of the ␮LCR-201A␥ construct extends to the ApaI site at position ⫺201, while in the ␮LCR-382A␥ and ␮LCR-730A␥ constructs the promoter extends to the StuI and SspI sites at position ⫺382 and ⫺730, respectively. The ␮LCR-1350A␥ construct previously described [32], was modified by generating a HindIII fragment containing the A␥ gene and 1350-bp of the promoter, followed by digesting with AseI or BstxI to release a 2772-bp HindIII-AseI fragment and a 2843-bp HindIII-BstxI fragment. These two fragments were cloned individually into the (Bluescript) pBS␮LCR plasmid by blunt-ended ligation to produce the ␮LCR-822A␥ and ␮LCR-893A␥ constructs. Stable transfections To establish the individual stable MEL cell clones, 10 ␮g of plasmid DNA from each of the seven constructs was linearized with ScaI and co-transfected by electroporation with 7 ␮g of pMC1Neo plasmid (Stratagene, LaJolla, CA) into 107 MEL cells growing in log phase as previously described [33]. Following electroporation the cells were suspended in Iscove’s modified Dulbecco medium containing 10% fetal bovine serum. After 48 hours, the transformed cells were placed in media containing 0.7 mg/mL of active G418. Limiting dilution assays were performed to obtain individual clones. Approximately 25–30 clones were used to establish each of the MEL cell pools analyzed in detail. Resistant clones were isolated 7 to 14 days after the addition of G418. Twenty-one pools (triplicates for each construct) containing the seven different constructs were established. The MEL cell pools were maintained in 10% fetal bovine serum, 0.9 mg/mL of active G418, and induced with either 2-mM NaB for 72 hours or grown in 60-mM ␣-ABA for 2 weeks. Erythroid differentiation was induced by growing the MEL cells in 3-mM hexamethyl bisacetamide (HMBA) for 72 hours. Approximately 80% of the MEL cells were benzidine positive at the time of harvest after HMBA induction. RNA analysis Total cytoplasmic RNA was prepared by the method of Chomcynski and Sacchi [34] and quantitated by UV spectrophotometry. Globin gene expression was analyzed by RNase protection with the following probes: pT7A␥ (170), linearized with BsteII, to give a 170-bp protected fragment, and pT7murine ␣ (128) linearized with HindIII to give a 128-bp protected fragment. The Tri-␤-actin probe (Ambion, Austin, TX) yielding a 245-bp protected fragment was used as an internal control for variations in RNA loading. RNA (200 ngs) was hybridized overnight at 45⬚C with 106 cpm of each radiolabeled probe. After digestion with RNase A, the protected fragments were separated on a 6% polyacrylamide-8M urea gel and autoradiographed. Human A␥, murine ␣, and murine actin mRNA was quantitated using a BioRad GS-250 phosphorimager (BioRad, Hercules, CA). Human A␥ globin mRNA levels were calculated as a percentage of murine ␣ corrected for gene copy number. Copy number determination Agarose plugs containing genomic DNA from MEL cells were prepared digested overnight with EcoRI followed by gel electrophoresis. DNA was transferred to a nylon membrane and hybridized by standard methods. Copy numbers were determined using 5⬘ hypersensitive site 3 (5⬘HS3) as a probe for the ␤ globin locus and murine Thy 1.1 as an internal diploid control. To ensure equal radioactive labeling of both probes, a 784-bp PstI 5⬘HS3 fragment

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was cloned into pW126, a construct containing a 544-bp BamHI Thy 1.1 cDNA fragment ligated into pBluescript (Stratagene) to produce pPN201. Digestion with EcoRI and HindIII released a 1.3-kb fragment containing both the 5⬘HS3 and Thy 1.1 fragments, that was labeled by random-priming using the Decaprime labeling system (Ambion) according to the vendor’s instructions. The ratio of 5⬘HS3 to Thy 1.1 obtained from phosphorimager quantitation was used to measure the difference in specific activity between the two fragments. The Thy 1.1 signal which represented two copies of the gene in the diploid genome was multiplied by the correction factor then divided by two to obtain a value for a single gene copy. Human transgene copy numbers were determined for each of the MEL cell pools by dividing the value obtained for the 5⬘HS3 signal by the corrected Thy 1.1 single-copy signal value. Transient transfections Six chloramphenicol acetyl transferase (CAT) reporter constructs were analyzed. The pCAT basic and the cytomegalovirus/CAT (pCMV/CAT) plasmids served as negative and positive controls, respectively, for CAT gene expression. In addition, four experimental plasmids were tested: the CAT reporter gene cloned downstream of the ⫺822 to ⫺893 sequence alone (pBRE/CAT), or in combination with either the CMV promoter (pBRE-CMV/CAT), or the minimal A␥ promoter from nucleotide ⫺201 to ⫹36 (pBRE-A␥/CAT). The fourth experimental plasmid contained the minimal A␥ promoter (pA␥/CAT) alone. The A␥ promoter fragments were generated using PCR amplification followed by direct sequencing. A ␤-galactosidase plasmid (2.5 ␮g) was cotransfected to control for variations in transfection efficiency. MEL cells (2.5⫻107) were grown in log phase in Iscove’s modified Dulbecco’s media and 10% fetal bovine serum, followed by electroporation with 20 ␮g of plasmid DNA at 260V and 960 ␮F using a BioRad Gene Pulser II system. After 8 hours, the cells were treated with NaB (2-mM), ␣-ABA (60-mM), or HMBA (3-mM) for 72 hours. Three to four independent transfections were performed for each of the six constructs tested. The CAT assay was performed using the Promega CAT Enzyme Assay system (Madison, WI) and 14 C-chloramphenical acetylation quantified using liquid scintillation per the vendor’s instructions. ␤-Galactosidase activity was measured at A420 using a Promega assay system. For pCAT assays representative values obtained were on the order of 40-cpm/␮L of extract/␤-galactosidase A420. The minimal detectable activity was 10-cpm/␮L of extract/␤-galactosidase A420 or fivefold over background under these assay conditions. Gel mobility shift assay Nuclear protein extraction was performed as previously described by Andrews and Faller [35]. In brief, approximately 5⫻106 cells were harvested, rinsed with phosphate buffered saline, and then resuspended in buffer A [10 mM HEPES pH7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride (Sigma Chemical Co., St. Louis, MO)]. The samples were incubated on ice until resuspended in buffer C (20 mM HEPES, pH7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride). Nuclear proteins were isolated from MEL cells in the uninduced, NaB-, or HMBA-induced states or from uninduced Helr cells and stored at ⫺70⬚C until used. Hela nuclear extract was purchased from Promega. Oligonucleotides dividing the ⫺822 to ⫺893 region into three fragments were analyzed. The se-

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quences for the sense strands used were: 5⬘BRE(⫺893 to ⫺856): 5⬘-TAATTCTAATCCACAGTACCTGCCAAAGAACATTCTAC3⬘; 3⬘BRE(⫺855 to ⫺822): 5⬘-CATCATCTTTACTGAGCATAGAAGAGCTACGCCA-3⬘ and BBRE(⫺865 to ⫺840): 5⬘-GAGCATCTACCATCATCTTTACCGA-3⬘. Gel mobility shift assays (GMSA) were performed with double stranded oligonucleotide probes end-labeled with [␥32P]-ATP in a T4-kinase reaction. Each gel shift experiment was performed with 4 ␮g of nuclear protein incubated with binding buffer [20% glycerol, 5 mM MgCl2, 2.5 mM EDTA, 250 mM NaCl, 50 mM Tris-HCl pH7.5] for 10 minutes at room temperature followed by incubation for an additional 20 minutes with the radiolabeled probe. For competition experiments excess cold competitor was preincubated with the nuclear extract before the radiolabeled probe was added. All binding reactions were performed in the presence of the nonspecific competitor poly(dI-dC) 0.25 ␮g/mL at a 50-fold excess. The following specific cold competitors were individually analyzed: Sp1(s): 5⬘-ATTCGATCGGGGCGGGGCGAGC-3⬘, Ap1(s): 5⬘-CGCTTGATGAGTCAGCCGGAA-3⬘, and Oct1(s): 5⬘-TGTCGAATGCAAATCACT- GAA-3⬘ (Promega), and p45 nuclear factor-erythroid 2 (p45NF-E2)(s): 5⬘-TGGGGAACCTGTGCTGAGTCACTGGAG3⬘ (Santa Cruz Biotechnology, Santa Cruz, CA). Protein-DNA complexes were resolved on a 4% nondenaturing polyacrylamide gel followed by autoradiography. Statistics Results of experimental points from multiple experiments were reported as the mean ⫾ standard error of the mean (SEM). Statistical analysis was performed using the two-tailed Student’s t-test for paired data. These tests were performed, and p values computed using SAS software (Statistical Analysis System, Cary, NC); p ⬍ 0.05 values were considered significant.

Results Truncation analysis to identify DNA sequences in the A␥ promoter that mediate butyrate inducibility Previous studies performed in transgenic mice carrying the human A␥ gene including 1350-bp of the promoter demonstrated ␥ gene induction by ␣-ABA [23,36]. To localize potential butyrate response elements in the A␥ promoter the recombinant fragments shown in Fig. 1A were used to establish stable MEL cell pools. Each construct consists of the same 2.5-kb ␮LCR cassette linked to the A␥ gene extending 3⬘ to nucleotide ⫹1950 relative to the cap site, with 5⬘ ends at the nucleotides shown in Fig. 1A. For each of the seven constructs, three MEL cell pools were established for a total of 21 independent pools analyzed in the uninduced state or in the presence of ␣-ABA, NaB, or HMBA. We chose to analyze cell pools to minimize the effects of variable gene expression levels that occur with individual MEL cell clones. ␣-ABA, in contrast to sodium butyrate, activates A␥ gene expression without inducing terminal differentiation [18]. Therefore, cells were cultured in the presence of ␣-ABA for 2 weeks before harvesting to analyze ␥-gene expression levels. HMBA, a known inducer of terminal differentiation in

MEL cells was used as a positive control. After HMBA (3mM) induction cell pellets were pink at the time of harvest and resulted in an average 27-fold increase in murine ␣ mRNA levels compared to actin mRNA levels. Following culture in the various agents total cellular RNA was prepared and analyzed by RNase protection assay. Copy numbers (Table 1) were determined for each MEL cell pool by multiple southern blot analyses. We observed some variability for murine ␣ gene mRNA production in the uninduced state for the different pools when compared with the actin control. For each pool analyzed ␥ mRNA was expressed as a percentage of murine ␣ mRNA, corrected for the number of copies of the transgene and endogenous murine ␣ genes. In the uninduced state low level A␥ gene expression was observed with the ␮LCR-141A␥ MEL cell pools (Fig. 1B, lane 1; Fig. 2A). Two peaks for basal A␥ gene expression in the absence of chemical induction was observed, one with the ␮LCR-201A␥ construct (␥ ⫽ 30.5% of murine ␣) and a second with the ␮LCR-730A␥ construct (␥ ⫽ 53.6% of murine ␣). These results suggest that the ⫺141 to ⫺201 as well as the ⫺382 to ⫺730 regions contain DNA sequence that interacts with positive acting transcription factors present in MEL cells that have not undergone terminal erythroid differentiation. MEL cells induced with HMBA showed increased human A␥ and murine ␣ gene expression; however, murine ␣ induction was significantly greater than that of human A␥. As a result the level of A␥ mRNA as a percent of murine ␣ mRNA decreased compared to uninduced cells (Fig. 2B). However, the HMBA-induced cells also displayed two peaks of A␥ gene activity with the ␮LCR-201A␥ and ␮LCR-730A␥ MEL cell pools. These results indicate that transcription factors present after MEL cells have undergone terminal differentiation interact with the DNA sequence between nucleotides ⫺141 and ⫺201 and nucleotides ⫺382 and ⫺730. For the uninduced MEL cell pools we observed a 17-fold increase in A␥ gene expression when the ␥ promoter was extended from nucleotide ⫺141 to ⫺201. In contrast, we observed a 27-fold increase in ␥ mRNA for the ␮LCR201A␥ construct in the presence of ␣-ABA (Fig. 2C). The main difference between the ␮LCR-141A␥ and ␮LCR201A␥ constructs is the presence of the “CACC” box in the latter. Increased A␥ gene expression for the ␮LCR-201A␥ construct compared with the ␮LCR-141A␥ construct suggests that the “CACC” may be involved in ␣-ABA inducibility. A second region mediating butyrate inducibility was observed for the ␮LCR-893A␥ MEL cell pools. This suggests a butyrate response element may be located between nucleotide ⫺822 and ⫺893 in the A␥ promoter (Fig. 1B, lanes 13–18; Fig. 2C). Although a small increase in A␥ gene activity was observed for the ␮LCR-893A␥ pools in the uninduced state, it was not statistically significant. How-

B.S. Pace et al./Experimental Hematology 28 (2000) 283–293 Table 1. ␥-Globin gene expression in uninduced MEL cell pools transfected with ␥ promoter truncation mutants Construct ␮LCR-141A␥

Pool No.

Copy No.

1 2 3

1.5 1.2 1.9 Mean ⫾ SEM 6.1 5.3 7.1 Mean ⫾ SEM 5.4 4.0 2.3 Mean ⫾ SEM 4.0 1.1 0.6 Mean ⫾ SEM 2.3 2.0 2.4 Mean ⫾ SEM 3.2 0.4 1.0 Mean ⫾ SEM 5.1 3.9 2.2 Mean ⫾ SEM

␮LCR-201A␥

1 2 3

␮LCR-382A␥

1 2 3

␮LCR-730A␥

1 2 3

␮LCR-822A␥

1 2 3

␮LCR-893A␥

1 2 3

␮LCR-1350A␥

1 2 3

%␥ⲐMu␣ mRNA per copy 1.5 2.4 1.6 1.8 ⫾ 4.39 19.6 31.8 40.2 30.5 ⫾ 5.91 20.2 36.8 11.5 22.8 ⫾ 7.44 68.8 58.0 34.2 53.6 ⫾ 10.21 30.5 17.2 21.1 20.1 ⫾ 1.90 30.5 14.1 25.3 23.3 ⫾ 4.76 5.5 4.7 5.9 5.4 ⫾ 1.23

The copy numbers for the MEL cell pools are shown. ␥ mRNA levels in the uninduced state were calculated as the %␥ⲐMu␣ mRNA per copy of the transgene and endogenous murine ␣ gene mean ⫾ standard error of the mean (SEM).

ever, in the presence of ␣-ABA there was a 5.5-fold increase (p ⬍ 0.05) in A␥ gene activity when the ⫺822 to ⫺893 sequence was present. The ␮LCR-1350A␥ construct showed very low A␥ gene expression in the uninduced MEL pools as well as in the presence of ␣-ABA or HMBA. Similar experiments were complete for NaB-induced MEL cell pools with a 30-fold increase in A␥ gene expression in the ␮LCR-201A␥ construct and a 6.5-fold increase in ␥ mRNA with the ␮LCR-893A␥ construct (Fig. 2D). These data support the presence of butyrate response elements in the proximal and distal ␥ promoter that respond to both NaB and ␣-ABA. The transcription factors gata-1, octamer-1 and Sp-1 are known to bind in the ⫺141 to ⫺201 region [1], therefore, butyrate may modify the binding activity for one of these nuclear proteins or a yet unidentified transcription factor. Likewise, the ⫺822 to ⫺893 ␥ promoter sequence was inducible by both NaB and ␣-ABA. Although these agents have different effects on cell growth in culture, they have similar patterns for A␥ gene activation in our system. The upstream ⫺822 to ⫺893 region has not

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been previously studied in details and may have functional significance and/or bind novel transcription factors. The ability of the ⫺822 to ⫺893 sequence to enhance transcription and the pattern for nuclear protein binding to this region was analyzed in detail. Functional analysis for the A␥ gene butyrate inducible elements Studies were performed to test the enhancing properties of the ⫺822 to ⫺893 ␥ promoter sequence (␥BRE) either alone or combined with the CMV or minimal ␥ promoter, in a transient assay system. Five recombinant fragments were cloned upstream of a CAT reporter gene and analyzed in the presence of ␣-ABA or NaB. Both agents have been shown to induce ␥ gene expression in vitro in primary erythroid progenitor [18] and in vivo in transgenic mice [31,36]. NaB is an inducer of terminal differentiation whereas ␣-ABA induces globin gene expression in the absence of terminal differentiation [18]. Therefore, in a transient assay system ␣-ABA might be predicted to be a less potent inducer of reporter gene activity due to requirements for a longer time in culture to obtain maximum effects. For the six reporters constructs analyzed in MEL cells (Fig. 3A) CAT activity was unchanged in the presence of ␣-ABA (60-mM) for 72 hours (data not shown). These findings are consistent with the inability of ␣-ABA to induce terminal differentiation and the limited time in culture in a transient assay system. In contrast, we observed an 8.5-fold (p ⬍ 0.05) increase in CAT activity for the control pCMV/ CAT plasmid in the presence of 2-mM NaB for 72 hours (Fig. 3B). CAT activity was increased 2.0-fold with the pBRE/CAT plasmid after NaB induction, levels similar to that obtained for pCAT (1.4-fold) suggesting the ␥BRE fragment is unable to serve as a promoter for CAT gene activation. The pA␥/CAT plasmid containing the minimal A␥ promoter (⫺201 to ⫹36) was induced 6.1-fold (p ⬍ 0.05) in the presence of NaB, levels comparable to that obtained for the pCMV/CAT positive control plasmid. Moreover, combining the ␥BRE with the minimal A␥ promoter resulted in an 11.5-fold increase in CAT activity (Fig. 3B) suggesting the ␥BRE interact with the minimal ␥ promoter to further enhance butyrate inducibility. In contrast, combining the ␥BRE with the heterologous CMV promoter (pBRE-CMV/ CAT) inhibited the 8.5-fold increase in CAT activity observed with NaB induction for the CMV promoter alone (Fig. 3B). Finally, in all of the reporter plasmids tested HMBA induction resulted in minimal increases in CAT activity compared to uninduced MEL cells. This suggest interactions between hypersensitive sites in the ␤ globin LCR and the minimal A␥ promoter may be necessary to facilitate CAT gene activation by HMBA in MEL cells. These data in conjunction with observations in the stable MEL cell pools support the presence of a functional butyrate response element between nucleotides ⫺822 and ⫺893.

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Figure 2. Globin mRNA levels (mean ⫾ SEM) for the stable MEL cell pools. The individual pools were tested in either the uninduced (white bars), hexamethyl bisacetamide-induced (HMBA, striped bars), ␣-ABA-induced (black bars), or NaB-induced (gray bars) states. (A) In the uninduced state increased baseline ␥ mRNA synthesis was observed for the ␮LCR-201A␥ and ␮LCR-730A␥ pools. (B) The positive control HMBA, induced terminal differentiation and increased ␥ globin and murine ␣ mRNA levels. Peak ␥ mRNA synthesis was also observed for the ␮LCR-201A␥ and ␮LCR-730A␥ pools. Note the lower values on the Y-axis for the HMBA-induced MEL cell pools. (C) ␣-ABA treatment resulted in a further increase in ␥ gene activity in the ␮LCR-201A␥ pool. A second inducible element was identified in the ␮LCR-893A␥ pool in the presence of ␣-ABA, suggesting the presence of a butyrate response element in this region. (D) Induction with NaB (2 mM) increased ␥ gene activity for the ␮LCR-201A␥ and ␮LCR-893A␥ pools comparable to the response produced by ␣-ABA.

Characterization of the protein binding pattern for the ⫺822 to ⫺893 A␥ promoter region The data obtained from the stable MEL cell pools and transient transfection experiments suggest a functional role for the ⫺141 to ⫺201 and ⫺822 to ⫺893 regions as mediators of butyrate inducibility. The DNA sequence between nucleotide ⫺822 and ⫺893 linked to the proximal A␥ promoter mediates increased CAT gene activity in the presence of NaB. McCaffrey et al. [29] demonstrated a role for the ␥ promoter distal CCAAT box and to a lesser extent the CACC box in butyrate inducibility. Several proteins are known to bind to the proximal ␥ promoter that may be involved in the mechanism for butyrate inducibility. Extensive in vivo and in vitro analysis by Ikuta et al. [30] showed changes in transcription factor binding in the proximal ␥ promoter in the presence of butyrate. In contrast, less is known about transcription factors binding in the upstream promoter region. Therefore, protein-binding studies were performed for the ⫺822 to ⫺893 region using gel mobility shift assays. We chose to analyze nuclear extracts from NaB- or HMBA-induced MEL cells given the ability of NaB to induce A␥ gene activity and stimulate CAT activity in our transient assay system.

Oligonucleotides that divide the ⫺822 to ⫺893 region into three fragments were analyzed. All binding reactions were performed with 4 ␮g of total nuclear proteins and the nonspecific competitor poly(dI-dC) at 50-fold excess. Nuclear extracts from MEL cells in the uninduced state produced a single specific protein-DNA complex with the 5⬘ BRE (⫺893 to ⫺856) probe (Fig. 4A, lanes 1 and 2). Induction with NaB markedly reduced binding for the B4 protein with the appearance of a second specific B1 complex (Fig. 4A, lanes 3 and 4) suggesting the B4 protein is negative-acting and the B1 protein is a positive-acting transcription factor. Studies performed with nuclear extract from HMBA-induced MEL cells also showed significant B4 protein binding (Fig. 4A, lane 5) comparable to uninduced MEL cell extract. For nonerythroid Hela cell extract two specific protein-DNA complexes were formed with the 5⬘BRE probe. The upper complex comigrated with B1 and the lowest complex migrated slower than the B4 complex. Gel shift analyses were also performed with the 3⬘BRE (⫺855 to ⫺822) A␥ promoter fragment. A single specific protein-DNA complex was obtained with uninduced MEL cell extract that was significantly increased with NaB and HMBA induction (Fig. 4B, lanes 1, 3, and 5). The pattern of

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Figure 3. Transient transfection experiments to determine the functional properties for the ⫺822 to ⫺893 A␥ promoter sequence. (A) A schematic of the six chloramphenicol acetyl transferase (CAT) reporter plasmids analyzed. The CAT gene and promoter fragments are not drawn to scale (see Material and Methods section). (B) CAT activity for the uninduced (white bars), NaB-induced (black bars), or HMBA-induced (striped bars) MEL cells. There was an 8.5-fold increase in CAT activity with NaB induction for the pCMV/CAT positive control plasmid. The pA␥/CAT plasmid was induced 6.1-fold in the presence of NaB; with the addition of the ⫺822 to ⫺893 (␥BRE) fragment to produce the pBREA␥/CAT plasmid we observed an 11.5-fold increase in CAT activity. In contrast, the ␥BRE fragment had a negative effect on the heterologous CMV promoter demonstrated by a loss of induction by NaB. The mean ⫾ SEM is shown for three to four independent experiments performed for each construct tested.

binding observed with NaB and HMBA induction is similar to that obtained with uninduced nuclear extract from human erythroleukemia cells (data not shown) which carry a fetal phenotype and actively express both the ␧ and ␥ globin genes [37]. The protein bound in the B2 complex increased with NaB- and HMBA-induced MEL cell and Hela nuclear extracts (data not shown), suggesting the B2 protein is ubiquitous and its binding to the 3⬘BRE can be modified by both NaB and HMBA. The third oligonucleotide analyzed BBRE (⫺865 to ⫺840) overlaps with the 5⬘BRE and 3⬘BRE DNA fragments. We observed two specific protein-DNA complexes (Fig. 4C, lanes 1 and 3) that were unchanged with NaB- or HMBA-induced nuclear extract suggesting these proteins are not involved in butyrate inducibility. Competition experiments to identify potential transcription factors in the major protein-DNA complexes were completed with Sp-1, Ap-1, NF-E2, and Oct-1 oligonucleotides at 50-fold excess. We observed an absence of competition for the B4 protein-DNA complex (5⬘BRE-B4) formed with uninduced MEL cell extract and the 5⬘BRE probe (Fig. 5, left panel). Similar results were obtained for the B2 protein-DNA complex (3⬘BRE-B2) formed with uninduced

and NaB-induced MEL cell extract and the 3⬘BRE probe. For both the 5⬘BRE and 3⬘BRE probes specific competition was obtained with the respective self cold oligonucleotides and with competitor sequences corresponding to the opposite half of the ⫺893 to ⫺822 sequence (Fig. 5, lanes 3 and 10). Cross competition between the 5⬘BRE and 3⬘BRE probes may be due to the AAGAGC, GAGCAT and GGTAAAGA (in the anti-sense direction in 3⬘BRE) motifs present in both fragments possibly serving as binding sites for transcription factors.

Discussion The control of ␥ gene activity during development involves interactions between cis active regulatory elements and stage-specific transcription factors [1]. In the adult, ␥ gene silencing may be accomplished through competition between the ␥- and ␤-globin promoters for interaction with the LCR [38–40] and/or interactions between the ␥ gene promoters and specific transcription factors that repress ␥ gene expression. Several ␥ promoter transcription factors have been identified. In particular, the stage selector protein that

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Figure 4. (A) Protein binding pattern for the ⫺822 to ⫺893 (␥BRE) promoter sequences. Single stranded oligonucleotides were annealed and end-labeled with [␥32P]-ATP for use in protein binding reactions (see Material and Methods section). (A) The 5⬘BRE (⫺893 to ⫺856) probe was analyzed with nuclear extracts from uninduced (U), NaB-induced (N), or HMBA-induced (H) MEL cells or from Hela (HE) cells in the absence (⫺) or presence (⫹) of cold competitor 5⬘BRE sequence. Note the appearance of B1 and the disappearance of the B4 protein complex with NaB-induced nuclear extract. (B) Nuclear extracts from NaB- or HMBA-induced MEL cells were analyzed with the 3⬘BRE (⫺855 to ⫺822) probe in the absence (⫺) or presence (⫹) of cold competitor 3⬘BRE sequence. Both NaB and HMBA induction resulted in increased binding for the B2 protein to the 3⬘BRE probe (lanes 1, 3, and 5). (C) The protein binding pattern for the bridging BBRE (⫺865 to ⫺840) A␥ sequence was analyzed. Similar experiments in the absence (⫺) or presence (⫹) of cold competitor BBRE sequence were completed. The binding pattern for the two specific DNA-protein complexes was unchanged with nuclear extract obtained after NaB induction (lanes 1 and 3). A third protein-DNA complex (B3) was observed with HMBA-induced and Hela nuclear extract (lanes 5 and 7).

binds to the stage selector element located in the ⫺50 ␥ region is thought to play an important role in activating ␥ gene expression during fetal-stage development [41]. A mechanism for ␥ gene reactivation after the switch has occurred is modulation of nuclear transacting factors that interact with DNA regulatory elements in the promoter. Limited data is available about the regulatory sequences in the ␥ gene through which fetal hemoglobin inducing agents such as butyrate exert their effects to reactivate gene expression. In this study we performed a detailed analysis of the A␥ promoter and identified DNA regulatory sequences inducible by butyric acid. Sodium butyrate inhibits the enzyme histone deacetylase altering histone acetylation levels and chromatin structure [22,42]; this could be a putative mechanism for gene activation. Studies performed with chicken erythrocyte mononucleosomes support a mechanism for globin activation that is independent of acetylation status such that “poised genes” as well as transcriptionally active genes carry similar histone acetylation levels [43]. It has been suggested that maximally acetylated histones represent the “ground state” of all poised

genes and when an inducible gene is stimulated, the level of histone acetylation does not change [44]. Glauber et al. [24] obtained similar results for the chicken ␳ gene by demonstrating no change in bulk histone acetylation levels in stably transfected MEL cells following NaB induction. Furthermore, they demonstrated that specific 5⬘ flanking DNA sequences are required for ␳ gene activation by butyrate [24]. In our study, seven stable MEL cells pools were used to demonstrate the presence of two regions with increased basal ␥ gene activity, one between nucleotides ⫺141 and ⫺201 and a second between nucleotides ⫺382 and ⫺730. In the presence of the proximal region there was a further increase in A␥ gene activity in response to ␣-ABA and butyrate treatment but these agents did not induce the ␮LCR730A␥ pool. Further upstream, a second butyrate inducible element was identified between nucleotide ⫺822 and ⫺893. An unexpected observation was no inducibility by either ␣-ABA or NaB for the ␮LCR-1350A␥ MEL cell pools compared with a sixfold induction in ␮LCR-1350A␥ transgenic mice treated with ␣-ABA. A possible explanation for these results is that changes in chromatin structure or in

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Figure 5. Competition experiments to identify transcription factors that bind to the ␥BRE. Competition reactions (50-fold excess cold competitor) with the 5⬘BRE and 3⬘BRE probes were completed with uninduced (MELu) and NaB-induced (MELn) MEL cell nuclear extract respectively. The competitors tested included the 5⬘BRE (5⬘), 3⬘BRE (3⬘), Sp-1 (S), Ap-1 (A), NF-E2 (Nf) and Oct-1 (O) (see Material and Methods section for sequences). Note specific competition for the major protein-DNA complex 5⬘BRE-B4 formed with 5⬘BRE probe and uninduced MEL cell extract (left panel, lanes 1 and 2). (The 5⬘BRE-B4 complex corresponds to the B4 complex demonstrated in Fig. 4A, lane 1). Similarly, the 3⬘BRE-B2 complex formed with uninduced and NaB-induced MEL cell nuclear extract and the 3⬘BRE probe (Fig. 4B, lanes 1 and 3) was competed in a specific manner (right panel, lane 9). Competition experiments performed with competitor sequence from the opposite half of the ␥BRE sequence (lane 3 and 10) demonstrated cross-competition and similarities between the 5⬘BRE and 3⬘BRE fragments.

DNA binding factors elicited by ␣-ABA in MEL cells do not mimic exactly the changes elicited by this agent in primary cells in vivo. Alternatively, a negative element may be present between position ⫺893 and ⫺1350 that is active in MEL cells but inactive in primary cells. These findings raise the question of physiologic significance for the ␥BRE sequence. The data obtained with CAT assays support a functional role for the ␥BRE and GMSA demonstrated transcription factor binding to the ␥BRE. Ultimately butyrate inducibility in ␮LCR-893A␥ transgenic mice would confirm physiologic relevance for the ⫺822 to ⫺893 region. In another model system, Yuan and coworkers [26] identified butyrate inducible elements including the transactivation response region in the long terminal repeat of the human immunodeficiency virus type 1 gene. Data obtained in our stable MEL cell system suggests that Sp-1 binding to the CACC box may play a role in ␥ gene NaB inducibilty. Previous data obtained from transient expression experiments in K562 cells demonstrated that butyrate can act in the ␥ promoter through the distal “CCAAT” box [45], the stage selector element [30], DNA regulatory sequences in the proximal promoter [46], or the ␤ globin LCR [40]. McCaffrey et al. [29] demonstrated that the distal CCAAT box is required for response to trapoxin and trichostatin, two specific inhibitors of histone deacetylase. These results support a role for both histone deacetylase inhibition and transcription factor modification in ␥ gene activation in the presence of butyrate. We previously demonstrated a twofold increase in A␥ gene activity following ␣-ABA treatment in transgenic mice carrying the minimal ␥ promoter versus a sixfold in-

crease when the ␥ promoter was extended further upstream to position ⫺1350 which includes the ⫺822 to ⫺893 sequence [31]. In our transient assay system ␣-ABA failed to induce the constructs tested. Other investigators have demonstrated differences in the ability of ␣-ABA and NaB to induce terminal differentiation in culture [18]. This may account for the observed differences in ␥ promoter inducibility for ␣-ABA and NaB in our transient assays. In contrast, in the presence of NaB, we observed induction for the pA␥⵮ CAT and pBREA␥CAT constructs. When the ⫺822 to ⫺893 sequence was analyzed alone there was no increased promoter activity in response to NaB. Similar requirements for proximal promoter elements have been demonstrated for butyrate response elements identified in the upstream conserved element in the histone H1 gene promoter [25]. Khochbin and Wolffe [25] demonstrated that butyrate-induced transcription requires nucleoprotein interactions both in the upstream conserved element and more proximal promoter regulatory sequences. The data presented in this study supports the ⫺822 to ⫺893 region as a DNA regulatory region that contributes to ␥ gene butyrate inducibility. Whether the ␥BRE is specific for the ␥ promoter or can stimulate other globin promoters, i.e., ␤ gobin, will be tested experimentally. Several investigators have demonstrated transcription factor modification as a mechanism for gene activation by butyrate. Both acetylation and phosphorylation of nuclear proteins that bind to specific DNA sequences occur in cells treated with NaB [47]. More recently, it has been shown that NaB induces tyrosine phosphorylation and activation of MAP kinase [48]. Studies by Ikuta et al. [30] demonstrated increased ␣CP2 binding in the ␥ promoter after NaB induc-

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tion. ␣CP2 forms a heterodimer in the stage selector protein complex that is important for ␥ gene expression during fetal-stage development. Similarly, in this study protein-binding experiments demonstrated protein interactions in the ⫺822 to ⫺893 region that were modified in the presence of NaB. Decreased B4 protein binding observed with the 5⬘BRE probe and NaB-induced MEL cell extract, suggest that proteins in this complex are repressors of A␥ gene activity during adult-stage erythroid growth. The binding pattern for 3⬘BRE-B2 suggest a positive-acting transcription factor may be bound at increased levels in the presence of NaB. The upstream ␥BRE identified in our studies was not demonstrated by McCaffrey et al. [29] in K562 cells. This suggests the 5⬘BRE-B4 protein observed with uninduced MEL cell extract may be bound at higher levels during adult-stage development and silence ␥ gene expression. Likewise, one might speculate that there might be a requirement for high levels of the positive-acting factor 3⬘BRE-B2 to induce ␥ gene activity. This interpretation may explain the lack of butyrate inducibility observed by McCaffrey et al. [29] in K562 cells that have a fetal phenotype. Changes in the level or type of nuclear transcription factors play an important role in the control of sequential globin gene expression in each developmental stage [1]. Isolation and characterization of the specific transcription factors involved in gene activation by butyrate will help clarify the role of these proteins in the control of ␥ gene expression. Acknowledgments This study was supported by a grant from the American Heart Association, Alabama Affiliate, Inc. Grant# AL-G-950023, and an NIH Research Training Grant# HL03396. We want to thank Cindy Anderson for assisting with manuscript preparation, and Amy Ferry and Carlos Monteiro for technical assistance.

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